Functional Characterization of IL-17F as a Selective Neutrophil Attractant in Psoriasis  Hideaki Watanabe, Mio Kawaguchi, Sawa Fujishima, Miyoko Ogura, Satoshi Matsukura, Hiroko Takeuchi, Motoi Ohba, Hirohiko Sueki, Fumio Kokubu, Nobuyuki Hizawa, Mitsuru Adachi, Shau-Ku Huang, Masafumi Iijima  Journal of Investigative Dermatology  Volume 129, Issue 3, Pages 650-656 (March 2009) DOI: 10.1038/jid.2008.294 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 IL-17F induces IL-8 production by NHEKs. (a) IL-8 gene expression by IL-17F in NHEKs. Total RNA was extracted from the cell lysates 4 and 24hours after stimulation with 100ngml−1 IL-17F, 100ngml−1 TNF-α, 100ngml−1 IL-17A, and PBS control. The levels of mRNA for IL-8 were calculated as the fold induction compared with the PBS control using real-time PCR. (b) Analysis of IL-8 production in NHEKs stimulated with IL-17F (100ngml−1), TNF-α (100ngml−1), IL-17A (100ngml−1), and PBS control. IL-8 protein release in the supernatant was determined by ELISA as described in “Materials and Methods”. Results represent mean±SEM from at least three independent experiments. *P<0.05 was considered significant. Journal of Investigative Dermatology 2009 129, 650-656DOI: (10.1038/jid.2008.294) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 IL-8 gene expression in the mouse skin induced by IL-17F. Ear tissue from the IL-17F-treated and PBS control mice were collected 24hours following intradermal injection and total RNA was isolated from each ear specimen. The primer sequence was as follows: upstream 5′-ATGGCTGGGATTCACCTCAA-3′, downstream 5′-AAGCCTCGCGACCATTCTT-3′. The expression of IL-8 mRNA 24hours was 2.9-fold higher than that in the control group. Results represent mean±SEM from three independent experiments. Journal of Investigative Dermatology 2009 129, 650-656DOI: (10.1038/jid.2008.294) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of inhibitors on IL-8 protein production in NHEKs. The cells were preincubated with varying concentrations of PD98059, U0126, SB202190, SP600125, or Me2SO vehicle for 1hour, followed by stimulation with IL-17F (100ngml−1) for 24hours. Results represent mean±SEM from three independent experiments. *P<0.05 was considered significant versus IL-17F-stimulated cells without addition of inhibitors. Journal of Investigative Dermatology 2009 129, 650-656DOI: (10.1038/jid.2008.294) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Treatment with IL-17F induces ERK1/2 phosphorylation in the mouse skin. Ear tissue samples collected at 0.5, 1, and 2hours following IL-17F injection (100ng in 50μl of PBS) were prepared. Western blot analysis of phosphorylated and total ERK1/2 was performed on these skin samples. The activation of ERK1/2 was seen at 0.5, 1, and 2hours after injection. The results shown are representative of three separate experiments. Journal of Investigative Dermatology 2009 129, 650-656DOI: (10.1038/jid.2008.294) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Histologic examination. Histopathological findings 48hours after intradermal injection of (a) IL-17F, (c) TNF-α, (d) IL-17A, and (e) PBS control. Bar=100μm. (b) High magnification of a. Infiltrating cells in the dermis consisted mainly of neutrophils (arrow). Bar=50μm. (g) A cell count was performed as described in “Materials and Methods”, and the density of dermal infiltration was expressed as mononuclear cells, neutrophils, eosinophils, and total number of cells. No infiltrating eosinophils were seen in both the IL-17F-treated and other groups. The number of neutrophils in the dermis was significantly increased by injection of recombinant mouse IL-17F compared with the control group (*P<0.05) and the inflammatory cells in the dermis were significantly but not completely blocked by anti-IL-8 antibody (*P<0.05) (f, g). Bar=100μm. Journal of Investigative Dermatology 2009 129, 650-656DOI: (10.1038/jid.2008.294) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 IL-17F protein in the skin of psoriasis patients. (a) Representative results from analysis of IL-17F protein production in the nonlesional and lesional skin of psoriasis patients. Skin specimens were homogenized and the supernatants were examined by western blotting. (b) Protein level of IL-17F in the psoriatic lesional skin was higher than in nonlesional psoriatic skin (*P<0.05). Low level of IL-17F protein was also detected in nonlesional psoriatic skin. The results were expressed as the mean±SEM (n=5). *P<0.05 was considered significant. Journal of Investigative Dermatology 2009 129, 650-656DOI: (10.1038/jid.2008.294) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions